Genome binding/occupancy profiling by high throughput sequencing
Summary
Chromatin modifications and the promoter associated epigenome are thought to be important for the regulation of gene expression. However, the mechanisms by which chromatin modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed target gene promoters through CFP1 which engages in a unique form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed suggesting that the normal targeting and function of SET1 complex is central to creating an appropriately functioning vertebrate promoter associated epigenome.
Overall design
CFP1 fl/fl mouse embryonic stem cells were treated with tamoxifen (TAM) for 96h to ablate CFP1 protein levels. ChIP-seq experiments were performed to determine the genome-wide occupancy of CFP1 and SET1A, the catalytic subunit of the SET1 complex. Since SET1A occupancy is altered upon deletion of CFP1, we used a calibrated ChIP-seq approach to assess the changes in H3K4me3 in this cell line. Moreover, we generated additional ES cell lines with CFP1 fl/fl background expressing exogenous GFP-tagged forms of CFP1 to analysed the effects of different CFP1 mutantions on H3K4me3 profile. We generated a GFP control line (EV), a wild type CFP1 line, a PHD and CxxC domains (PC) double point mutant line, and a SET1 interaction domain (SID) mutant line. Calibrated experiments were performed when possible mixing mouse cells with a Drosophila S2 line (SG4) prior to chromatin extraction.
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